Blood contains various lineages of blood cells having biological functions, such as the erythrocytic lineage associated with oxygen delivery, the megakaryocytic lineage generating thrombocytes, the granulocytic lineage associated with prevention of infections, the myeloid lineage such as monocytes and/or macrophages and the lymphocytic lineage responsible for immunity such as T cells and B cells. All these blood cells differentiate and mature from the common origin, hematopoietic stem cells, and are maintained and generated in an individual throughout its life. Hematopoietic stem cells are defined as cells having both pluripotency which allows them to differentiate into functional cells such as lymphocytes, erythrocytes and leukocytes and the ability to regenerate themselves while maintaining the pluripotency (self-renewal).
Previous studies have revealed that hematopoietic stem cells first diverge two ways into the myeloid lineage and the lymphoid lineage, then differentiate into myeloid stem cells (mixed colony forming cells, CFU-GEMM) and into lymphoid stem cells, respectively. Further, myeloid stem cells differentiate into erythrocytes via erythroid burst forming cells (BFU-E) and erythroid colony forming cells (CFU-E), into thrombocytes via megakaryocyte colony forming cells (CFU-MEG), into monocytes, neutrophils and basophils via granulocyte-macrophage colony forming cells (CFU-GM), and into eosinophils via eosinophil colony forming cells (CFU-EO), while lymphoid stem cells differentiate into T cells via T lymphoid progenitor cells and into B cells via B lymphoid progenitor cells. Among them, cells forming pluripotential colonies with diameters of at least 1 mm are called HPP-CFU colony forming cells and are known as the least differentiated hematopoietic progenitor cells, similarly to mixed colony forming cells (CFU-GEMM). These myeloid stem cells and various hematopoietic progenitor cells derived from them are identified by the properties of colonies they form on soft agar, semisolid methylcellulose media or the like in the presence of various cytokines (Non-Patent Document 1).
In recent years, as a curative therapy for a number of intractable diseases such as various blood diseases attributed to hematopoietic dysfunction and immune dysfunction, cancer, immunodeficiency, autoimmune diseases and inborn error of metabolism, autologous or allogeneic transplantation of hematopoietic stem cells have been carried out. Quite recently, the effectiveness of hematopoietic stem cell transplantation in treating cerebral infarction, myocardial infarction and obstructive arteriosclerosis was reported (Non-Patent Documents 2, 3 and 4). Among them, bone marrow transplantation has been used in many cases of treatment and most established as a standard hematopoietic cell transplantation therapy. However, because for bone marrow transplantation, the human leukocyte antigens (HLA) of the bone marrow donor and the transplant recipient have to match closely, there is a problem that bone marrow from donors are in short supply. Besides, the need for at least 4 days of hospitalization and pain, fever and bleeding caused by collection of a large amount of bone marrow are a heavy burden to donors.
In addition to bone marrow, peripheral blood is also used as an alternative source of hematopoietic stem cells nowadays. Hematopoietic stem cells mobilized from the bone marrow to peripheral blood by administration of granulocyte colony stimulating factor (G-CSF) to a human are used for transplantation after enrichment using a blood cell separator. However, donors for peripheral blood hematopoietic stem cell transplantation have to bear a heavy burden of the need for administration of G-CSF for 4 to 6 consecutive days which may cause side effects (such as blood coagulation and spleen hypertrophy). Besides, because the efficiency of the mobilization of hematopoietic stem cells from the bone marrow to peripheral blood by G-CSF varies from donor to donor, hematopoietic stem cells are not obtained sufficiently in some cases.
Just recently, it was found that cord blood contains as many hematopoietic stem cells as bone marrow and is useful for hematopoietic stem cell transplantation (Non-Patent Document 5). Because cord blood transplantation does not require complete HLA matching and is less likely to cause severe acute graft-versus-host disease (GVHD) than bone marrow and peripheral blood transplantation, cord blood is established as useful and has been used more frequently. However, because cord blood is obtained in a small amount from one donor and does not contain many hematopoietic stem cells, its use is mainly limited to children.
Furthermore, hematopoietic stem cells are also considered as useful cells for gene therapy of fatal genetic diseases with no effective cure, HIV infection, chronic granulomatosis and germ cell tumor. However, in order to transfect hematopoietic stem cells with a retrovirus vector carrying a target gene efficiently, it is necessary to artificially grow hematopoietic stem cells, which are usually in the stationary phase, by releasing them into the cell cycle. Besides, there is a problem that for long-lasting expression of a transgene, the transfected hematopoietic stem cells have to be kept undifferentiated in culture. Therefore, a cell expansion method for efficient gene transfer has been demanded (Non-Patent Document 6).
To solve the above-mentioned problems with hematopoietic stem cell transplantation and gene therapy, a technique for expanding hematopoietic stem cells and/or hematopoietic progenitor cells ex vivo is demanded, and various culture methods have been attempted so far.
Here, hematopoietic stem cells and hematopoietic progenitor cells, which are to be cultured, are explained. It was revealed that in human, hematopoietic stem cells and various hematopoietic progenitor cells derived from them are found in populations of CD34+ cells expressing the CD34 molecule as a cell surface antigen, and hence hematopoietic stem cells can be enriched as a CD34+ cell population (Non-Patent Document 7). Specifically speaking, they are often enriched by mixing a cell population to be separated with a CD34 antibody labeled with magnetic beads and magnetically collecting CD34+ cells (Non-Patent Documents 8 and 9). CD34+ cell populations contain less than 10% of CD34+CD38− cell populations not expressing the CD38 molecule as a cell surface antigen. It has come to be considered that hematopoietic stem cells are more enriched in CD34+CD38− cell populations than in CD34+ cell populations (Non-Patent Documents 10 and 11). In order to determine the proportion of undifferentiated hematopoietic progenitor cells in a cell population, HPP-CFU colony forming cells are usually counted as mentioned above (Non-Patent Document 12). In recent years, it has become possible to experimentally assay human hematopoietic stem cells for bone marrow repopulating ability by using NOD/SCID mice obtained by crossing diabetic mice and immunodeficient mice. The cells detected by this assay are called SCID-repopulating cells (SRC) and considered the closest to human hematopoietic stem cells (Non-Patent Document 13).
Conventional techniques for expanding hematopoietic stem cells and/or hematopoietic progenitor cells will also be explained. As mentioned above, since hematopoietic stem cells are more enriched in CD34+ cells, CD34+ cells are mainly used as the starting cells for expansion. Expansion of hematopoietic stem cells and is hematopoietic progenitor cells from CD34+ cells in culture in the presence of a cytokine or a growth factor such as stem cell factor (SCF), interleukin 3 (1-3), interleukin 6 (IL-6), interleukin 6 (IL-6)/soluble IL-6 receptor complex, interleukin 11 (IL-11), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), flk2/flt3 ligand (FL), thrombopoietin (TPO) and erythropoietin or Notch ligand (such as Delta 1) has been reported (Patent Documents 1, 2 and Non-Patent Documents 12, 14 and 15). Among them, TPO is especially excellent in hematopoietic stem cell expansion effect and used for in most of cases of expansion (Non-Patent Document 16). Hematopoietic stem cells and hematopoietic progenitor cells expand in culture in the presence of such various cytokines and growth factors, but hematopoietic stem cells expand only by several times. Besides, these cytokines and growth factors are all produced as recombinant proteins, it may be difficult to obtain them for expansion stably in a large amount at low cost quickly.
For ex vivo expansion of hematopoietic stem cells, coculture systems using a different type of cells as feeder cells in the presence of various cytokines were reported. For example, expansion of hematopoietic stem cells in coculture with human bone marrow stromal cells was attempted (Non-Patent Document 17). An attempt to expand CD34+ cells in the presence of TPO, FL and SCF using mouse bone marrow cell line HESS-5 was also reported (Non-Patent Document 18). However, these coculture systems use foreign cells, there is a risk that cells infected with an unknown pathogen whose existence has not been confirmed may also be transplanted to patients. Furthermore, when stromal cells from a different kind of animal are used, the stromal cells have to be separated completely from CD34+ cells because otherwise there is a risk of causing immune response in the recipient after transplantation.
In addition, ex vivo expansion of hematopoietic stem cells in culture in the presence of various cytokines such as TPO combined with low molecular weight compounds, not just various cytokines only, has been reported. Examples of such low molecular weight compounds include copper chelators, the combination of a histone deacetylase inhibitor and a DNA methylase inhibitor, all-trans retinoic acid, aldehyde dehydrogenase inhibitors (Non-Patent Documents 19, 20 and 21 and Patent Document 3). However, addition of any of them is not effective enough since hematopoietic stem cells expand by only several times, or cells have to be cultured for about 3 weeks.    Patent Document 1: JP-A-2001-161350    Patent Document 2: JP-A-2000-23674    Patent Document 3: JP-A-2002-502617    Non-Patent Document 1: Lu, L. et al.; Exp. Hematol., 11, 721-9, 1983    Non-Patent Document 2: Taguchi, A et al.; J Clin Invest., 114, 330-8. 2004    Non-Patent Document 3: Orlic, D et al.; Nature, 410, 701-5. 2001    Non-Patent Document 4: Tateishi-Yuyama, E et al.; Lancet, 360, 427-35. 2002    Non-Patent Document 5: Kurtzbert, J. et al.; New Eng. J. Med., 335, 157-66, 1996    Non-Patent Document 6: Nathwani, A C. et al.; Br J. Haematol., 128, 3-17, 2005    Non-Patent Document 7: Ema, H. et al.; Blood, 75, 1941-6, 1990    Non-Patent Document 8: Ishizawa, L. et al.; J Hematother., 2, 333-8, 1993    Non-Patent Document 9: Cassel, A. et al.; Exp. Hematol., 21, 585-91, 1993    Non-Patent Document 10: Bhatia, M. et al.; Proc. Natl. Acad. Sci. USA 94:5320-25, 1997    Non-Patent Document 11: Larochelle, A. et al.; Nat. Med., 2, 1329-37, 1996    Non-Patent Document 12: Shah, A J et al.; Blood., 87, 3563-3570, 2000    Non-Patent Document 13: Dick, J E et al.; Stem Cells., 15, 199-203, 1997    Non-Patent Document 14: Suzuki, T et al.; Stem Cells., 24, 2456-2465, 2006    Non-Patent Document 15: McNiece et al., Blood.; 96, 3001-3007, 2000    Non-Patent Document 16: Kaushansky, K et al.; Ann NY Acad Sci., 1044, 139-141, 2005    Non-Patent Document 17: Kuwano, Y et al.; Exp Hematol., 34, 150-8, 2006    Non-Patent Document 18: Kawada, H et al.; Exp Hematol., 5, 904-15, 1999    Non-Patent Document 19: Chute, J P et al.; Proc Natl Acad Sci USA., 103, 11707-12, 2006    Non-Patent Document 20: Milhem, M et al.; Blood., 103, 4102-10, 2004    Non-Patent Document 21: Leung, A Y et al.; Exp Hematol., 33, 422-7, 2005